Project 4/Summary NUP98-fusion oncogenes (eg, NUP98-HOXA9, -KDM5A & -NSD1) are associated with several pediatric hematological malignancies (eg, AML; 6-10%; and AEL; 20%) characterized by poor prognosis. All of these oncogenes fuse the N-terminal FG-repeat domain of the nucleoporin NUP98 to chromatin binding domains from a variety of transcriptional and epigenetic regulators (e.g., the histone binding PHD3 domain of KDM5A). Endogenous NUP98 primarily localizes to the nuclear pore complex, with its intrinsically disordered FG-repeat domain (~500 residues) filling the central pore. In contrast, NUP98 fusion oncoproteins (FOs) enter the nucleus, bind to chromatin and cause the formation of numerous, sub-micron sized nuclear puncta. Importantly, these puncta recruit epigenetic and transcriptional regulators to developmental regulatory genes, notably the HOX family, activate aberrant gene expression, and transform hematopoietic cells. Leukemias harboring NUP98 FOs are treated with standard AML-based remission induction therapy, with intensification/transplantation for suboptimally responsive disease, but prognosis is generally poor. Therefore, there is great need for new therapeutic strategies for treatment of NUP98 FO-driven leukemias. While the N- terminal domain of NUP98 FOs is considered undruggable because it is intrinsically disordered, their folded C- terminal domains offer opportunities for therapeutic development. For example, binding of the PHD3 domain within the NUP98-KDM5A FO to di-and tri-methylated lysine 4 of Histone 3 (H3K4-Me2/3) is required for rewiring of the epigenetic state of hematopoietic cells and their transformation. Further, deletion of the HOXA9 DNA binding domain from the NUP98-HOXA9 FO alters nuclear puncta formation and prevents cell transformation. Thus, therapeutic strategies targeting the interactions of these folded domains with chromatin have potential to inhibit cell transformation by NUP98 FOs. Here, we propose to develop small molecules that bind the KDM5A PHD3 domain and inhibit its binding to H3K4-Me2/3 (Aims 1 & 2). We further propose to adapt PHD3 domain binders and inhibitors using the PROTAC (PROteolysis-TArgeting Chimaera) strategy to induce degradation of NUP98-KDM5A in cells (Aim 2). The inhibitors and PROTACs we develop will be tested against murine and human cellular models, and in vivo murine models of NUP98-KDM5A-driven AML.